In the past, lipoprotein metabolism has been monitored in vivo by introducing intravenously lipoproteins labelled with the radioisotope .sup.125 I, and following the radioactivity with time in blood and/or urine. Since the isotope is a gamma emitter, the radiation image depicts the sites of metabolic activity and to some extent the degree thereof. Lipoprotein metabolism is of special interest because of its close association with atherosclerosis. For example, as set forth in U.S. Application serial number 425,187, filed Sept. 18, 1982, atherosclerotic lesions take up .sup.125 I-labelled low density lipoproteins, and these lesions can be monitored by extracorporeal gamma radiation imaging in animals and man after introduction of the lipoproteins in the bloodstream. However, these measurements are somewhat limited by the poor imaging characteristics of .sup.125 I.
Isotopes, such as Technetium-99m, are often used in nuclear medicine because their short half life and high-energy gamma emmission provide for excellent external imaging while permitting in vivo absorption of only a low dose of radioactivity. However, coupling these isotopes to lipoproteins (and to biological molecules in general) may result in loss of native structure or biological function of the coupled molecules. Lipoproteins are especially unstable in that they readily denature at pH 7 and below. This restricts the range of chemical procedures that can be used to couple radioisotopes to lipoproteins. Furthermore, biological molecules also have "active sites" (parts of their molecules that are responsible for their biological interactions with other body chemicals or structural components), and attachment of a radioisotope to these sites may interfere chemically or sterically with these interactions. Therefore, even when an isotope-coupled molecule is not denatured, it is not necessarily biologically active in the same way and to the same degree as its non-coupled counterparts. Such an isotope-coupled molecule would not suffice for studying a particular biological reaction of interest.
Technetium-99m(.sup.99m Tc), is an isotope especially suited for external imaging. For study of technetium chemistry, the more stable isotope, 99Tc, is often used for convenience. This was demonstrated by Jones et al (Journal of Nuclear Medicine 21:279-281, 1980) and DePamphilis et al (Inorganic Chemistry 22:2292-2297, 1983) to form stable technetium-dithiolate chelates by reduction of pertechnetate (TcO.sub.4.sup.--). A reducing agent effective in making this stable complex is sodium dithionite. While the yield was not maximal at pH 11 or higher, some stable technetium-dithiolate chelates were formed at physiologic pHs (pH 5-8). There was no suggestion in those studies that technetium would form a stable complex with proteins and lipoproteins.
An abstract published by Steigman (Journal of Nuclear Medicine 16:573, 1975) discloses a technetium-horse serum albumin (HSA) complex prepared by reduction of pertechnetate with ascorbic acid and ferric chloride, and subsequent attachment to HSA through a series of steps in pH environments ranging from 9 to 2. These low pHs would denature lipoproteins. Accordingly, Steigman's procedure for complexing technetium to albumin could not be applied to radiolabel lipoproteins.
Many radioisotopes that have good imaging characteristics are known to chelate diethylene triamine pentaacetic acid (DTPA), a bifunctional chelating agent which can be covalently bound to amine groups of proteins. DTPA has been coupled to albumin (Krejarek and Tucker, Biochemical and Biophysical Research Communications 77:581-585, 1977; Hnatowitch et al, International Journal of Applied Radiation and Isotopes 33:327-332, 1982), and to immunoglogulin G (IgG) (Hnatowitch et al, Science 220:613-615, 1983), and the coupled product has been chelated with indium. DTPA is known to chelate other metals such as Tc, Pb, Hg, Ga, and Mn.
Krejarek and Tucker couple DTPA to HSA utilizing a carboxycarbonic anhydride of DTPA. The coupling procedure involves an initial reaction at pH 7-8, then dialysis against acetate buffer at pH 5.5, and subsequent isolation of the DTPA-HSA product by column chromatography at pH 3.5. Indium is added at pH 3.5 to chelate DTPA-HSA. The pHs employed by Krejarek and Tucker would also denature lipoproteins.
Hnatowitch uses a cyclic anhydride of DTPA, which appears to be more reactive with some proteins than the carboxycarbonic anhydride of DTPA, and therefore can be coupled to these proteins with greater efficiency and under milder reaction conditions. Optimal coupling occurs at pH 7 (again, a pH which may not be useable with lipoproteins).
Thus, both DTPA coupling procedures described above by Krejarek and Hnatowitch are not directly applicable to lipoproteins for subsequent chelation with a radioisotope. Even slight changes in reaction conditions are known to affect the resulting product. For example, altering reaction conditions can lower the efficiency of DTPA coupling to protein, which reduces the amount of radiolabel that can be chelated to the DTPA-coupled protein. The resulting product may have insufficient specific radioactivity for practical use. Also, as with attachment of any isotope to a biological molecule, the resulting product may not have the desired biological activity. Such changes in biological activity are even more likely to occur when larger compounds, such as DTPA, are coupled to the biomolecule.